Footprinting with an Automated Capillary DNA Sequencer

Wandee Yindeeyoungyeon, Mark A. Schell
2000 BioTechniques  
Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving( i ) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; ( ii ) brief exposure of the fragment to a DNA-binding protein and then DNase I; ( iii )
more » ... ; ( iii ) spin-column purification; and ( iv ) analysis of partial digestion products on the ABI P RISM ™ 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5 ′ untranslated region of xpsR , one of its regulated target genes. The advantages of this new method are that it ( i ) uses long-lived, safe and easyto-make fluorescently labeled target fragments; ( ii ) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and ( iii ) is cost effective.
doi:10.2144/00295st05 pmid:11084866 fatcat:qqep4f5ex5cqpluealla655pne